Genetic variability within Borrelia burgdorferi sensu lato genospecies established by PCR-single-strand conformation polymorphism analysis of the rrfA-rrlB intergenic spacer in ixodes ricinus ticks from the Czech Republic

Abstract

In Europe the Borrelia burgdorferi sensu lato complex is represented by five distinct genospecies: Borrelia burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii, Borrelia valaisiana, and Borrelia lusitaniae. These taxonomic entities are known to differ in their specific associations with vertebrate hosts and to provoke distinct clinical manifestations in human patients. However, exceptions to these rules have often been observed, indicating that strains belonging to a single genospecies may be more heterogeneous than expected. It is, therefore, important to develop alternative identification tools which are able to distinguish Borrelia strains not only at the specific level but also at the intraspecific level. DNA from a sample of 370 Ixodes ricinus ticks collected in the Czech Republic was analyzed by PCR for the presence of a approximately 230-bp fragment of the rrfA-rrlB intergenic spacer of Borrelia spp. A total of 20.5% of the ticks were found to be positive. The infecting genospecies were identified by analyzing the amplified products by the restriction fragment length polymorphism (RFLP) method with restriction enzyme MseI and by single-strand conformation polymorphism (SSCP) analysis. The two methods were compared, and PCR-SSCP analysis appeared to be a valuable tool for rapid identification of spirochetes at the intraspecific level, particularly when large samples are examined. Furthermore, by using PCR-SSCP analysis we identified a previously unknown Borrelia genotype, genotype I-77, which would have gone unnoticed if RFLP analysis alone had been used.

FIG. 1.

PCR-SSCP profiles of the intergenic spacers of B. valaisiana genotype I-52 (lane 2), B. garinii genotype J-21 (lane 3), a mixed infection with B. burgdorferi sensu stricto genotype I-208 and B. afzelii genotype D-3 (lane 4), and B. afzelii genotype D-3 (lane 5). Lane 1 contained a 10-bp ladder marker.

FIG. 2.

PCR-SSCP profiles of genotype I-77 (lane 1), B. afzelii genotype I-2 (lane 2), B. afzelii genotype D-3 (lanes 4, 5, and 7), and B. afzelii genotype G-32 (lane 8). Lanes 3 and 6 contained a 10-bp ladder marker.

FIG. 3.

Fifty percent majority rule consensus trees resulting from Bayesian analysis of an alignment of the intergenic spacer (269 bp) of representatives of the major Borrelia genospecies and our genotypes. GenBank accession numbers are indicated in parentheses. B. burgdorferi sensu stricto (B. burgdorferi s. str.) genotypes were used as outgroups. The thick lines indicate branches supported in ≥70% of the trees sampled. The reliability values shown at the nodes are the percentages of times that lineages occurred in the trees sampled.